Extracellular potential sensing element, device for measuring extracellular potential, apparatus for measuring extracellular potential and method of measuring extracellular potential by using the same

ABSTRACT

A sensing element for measuring extracellular potential including a substrate, a well provided in a substrate, a guide section provided on the wall of the well, and a detective electrode formed at a lower surface of the substrate. The guide section is for guiding drug. The well is provided at the bottom with a depression, and a first throughhole penetrating through the depression and the lower surface of the substrate. The well is for mixing a subject cell, a culture solution and the drug together. The above-configured sensing element accurately measures a change generated by a subject cell.

This application is a divisional of Ser. No. 10/991,269 filed Nov. 17,2004, now U.S. Pat. No. 7,396,673, issued Jul. 8, 2008, the entiredisclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an extracellular potential sensingelement and a device for measuring an extracellular potential, which areused for performing simply and quickly the electrophysiologicalevaluation of a biological sample, such as a cell, using anelectrochemical change generated by the biological sample as an index.The present invention relates also to an apparatus for measuringextracellular potential, and a method of measuring extracellularpotential by using the same.

2. Background Art

Drug screening has been conducted using the electrical activity of acell as an index. Conventionally, the electrical activity of a cell ismeasured by a patch clamp technique or a technique employing afluorescent pigment, or a light emitting indicator.

In a patch clamp technique, a small portion (hereinafter referred to as“patch”) of cell membrane is attached to a tip portion of amicropipette, and is used to electrically record with a microelectrodeprobe the ion transport through a single ion channel protein. The patchclamp technique is one of a few-number of cell biological techniqueswhich can be used to investigate the function of a single protein inreal time.

In the other technique, a light generated in response to a change in theconcentration of a particular ion is monitored employing a fluorescentpigment, or a light emitting indicator, for measuring the electricalactivity of a cell (hereinafter referred to as “fluorescence measuringtechnique”).

The patch clamp technique requires special techniques for preparation,manipulation and the like of a micropipette, and much time for measuringone sample. Therefore, the patch clamp technique is not suitable forscreening a large quantity of candidate compounds for a drug at highspeed.

The fluorescence measuring technique can screen a large quantity ofcandidate compounds for a drug at high speed. However, the fluorescencemeasuring technique requires a step of staining a cell. Duringmeasurement, pigments cause high background noise, and the fluorescenceintensity decreases with time, resulting in poor signal to noise ratio(S/N).

An alternative method has been disclosed in WO 02/055653 (hereinafterreferred to as “patent document 1”); that is a method of measuringextracellular potential (hereinafter referred to as “extracellularpotential measuring method”). The extracellular potential measuringmethod offers data of high quality level comparable to those by patchclamp technique. Furthermore, the extracellular potential measuringmethod can measure a large quantity of samples at high speed by a simpleprocess, as the fluorescence measuring technique does.

Patent document 1 discloses an extracellular potential measuring device(hereinafter referred to as “device”), which measures extracellularpotential or physicochemical change generated by a cell. The deviceincludes at least one well having means for holding a cell provided on asubstrate. The well has a sensing means for detecting an electricalsignal.

FIG. 40 illustrates the structure of a typified conventionalextracellular potential measuring device. Culture solution 110 is inwell 103. Subject cell (cell) 105 is captured or held by cell holdingsection 113 provided on substrate 102. Cell holding section 113 isformed of depression 104, opening section 106 and throughhole 107 insubstrate 102. Throughhole 107 is connected to depression 104 viaopening section 106. Detective electrode 109, or sensing means, isdisposed in throughhole 109, and detective electrode 109 is connectedwith a signal detection section (not shown) via wire 108.

During the measurement, cell 105 is sucked by a sucking pump (notshown), or the like means, from the throughhole 107 side, so that it isheld to be close to depression 104. At the same time, culture solution110 flows to the throughhole 107 side and makes contact with detectiveelectrode 109. Thus, an electrical signal generated as the result ofactivity of cell 105 is detected by detective electrode 109 disposed atthe throughhole 107 side, with no leakage into culture solution 110 inwell 103.

In the measuring method using a conventional extracellular potentialmeasuring device, cell 105 is reacted with drug (not shown). Therefore,drug needs to be injected into culture solution 110. The injection ofdrug into culture solution 110 inevitably causes a flow of culturesolution 110 in the neighborhood of cell 105. If the change in flow withculture solution 110 is substantial in the neighborhood of cell 105,fluctuation arises with culture solution 111 which is making contactwith detective electrode 109. The fluctuation arising in culturesolution 111 becomes noise to detecting by detective electrode 109.

In measuring the extracellular potential, the noise to detecting bydetective electrode 109 is a cause that deteriorates the S/N ratio of asignal detected by detective electrode 109. Noise is caused byfluctuation of culture solution 111 has a low frequency, approximately100 Hz or lower. While, the change in extracellular potential exhibitsDC signal or a signal that changes with a cycle of less thanapproximately 5 kHz. Namely, the two signals share an overlappingfrequency range.

It has been tried to reduce the noise by means of an electrical filteror the like, but it also cuts off the signal of low frequency regionclose to DC signal. In some cases, depending on the filtercharacteristics, even a higher frequency signal of 100 Hz is cut offeither. Consequently, it is difficult to detect accurately a signalwhich represents extracellular potential generated by cell 105.

SUMMARY OF THE INVENTION

A sensing element for measuring extracellular potential in accordancewith the present invention has a substrate, a well provided in thesubstrate, a guide section provided on the wall of the well, and adetective electrode disposed at a lower surface of the substrate. Theguide section is for guiding drug. The well is provided at the bottomwith a depression, and a first throughhole penetrating through thedepression and the lower surface of the substrate. The well is formixing a subject cell, a culture solution and the drug together.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a partially-cut-off and exploded perspective view showing anextracellular potential measuring device in accordance with a firstexemplary embodiment of the present invention.

FIG. 2 is a partially-cut-off perspective view showing a sensing elementused in the measuring device of FIG. 1.

FIG. 3 is a partially-cut-off and magnified perspective view showing akey portion of the sensing element of FIG. 2.

FIG. 4 is a cross sectional view used to show how to use the device ofFIG. 1.

FIG. 5 is a magnified cross sectional view showing the device of FIG. 1.

FIG. 6 is a magnified cross sectional view showing the device of FIG. 1.

FIG. 7 is a cross sectional view showing the device of FIG. 1.

FIG. 8 is a cross sectional view showing the device of FIG. 1.

FIG. 9 is a perspective view of a sensing element used in the device ofFIG. 1.

FIG. 10 is a perspective view of a sensing element used in the device ofFIG. 1.

FIG. 11 is a chart showing a flow of measuring extracellular potentialin the device of FIG. 1.

FIG. 12 is a characteristics chart of extracellular potential datameasured using the device of FIG. 1.

FIG. 13 is a cross sectional view used to show a method of manufacturingthe device of FIG. 1.

FIG. 14 is a partially magnified cross sectional view used to show amethod of manufacturing the device of FIG. 1.

FIG. 15 is a partially magnified cross sectional view used to show amethod of manufacturing the device of FIG. 1.

FIG. 16 is a cross sectional view used to show a method of manufacturingthe device of FIG. 1.

FIG. 17 is a cross sectional view used to show a method of manufacturingthe device of FIG. 1.

FIG. 18 is a cross sectional view used to show a method of manufacturingthe device of FIG. 1.

FIG. 19 is a cross sectional view used to show a method of manufacturingthe device of FIG. 1.

FIG. 20 is a cross sectional view used to show a method of manufacturingthe device of FIG. 1.

FIG. 21 is a cross sectional view used to show a method of manufacturingthe device of FIG. 1.

FIG. 22 is a partially-cut-off perspective view of an extracellularpotential measuring device in accordance with a second exemplaryembodiment.

FIG. 23 is a partially-cut-off perspective view showing the device ofFIG. 22.

FIG. 24 is a partially-cut-off perspective view showing a sensingelement used in the device of FIG. 22.

FIG. 25 is a partially-cut-off and magnified perspective view showing akey portion of the sensing element of FIG. 24.

FIG. 26 is a partially-cut-off perspective view showing an extracellularpotential measuring device in the present invention.

FIG. 27 is a partially-cut-off perspective view showing an extracellularpotential measuring device in the present invention.

FIG. 28 is a partially magnified cross sectional view used to show amethod of manufacturing the device of FIG. 22.

FIG. 29 is a partially magnified cross sectional view used to show amethod of manufacturing the device of FIG. 22.

FIG. 30 is a partially magnified cross sectional view used to show amethod of manufacturing the device of FIG. 22.

FIG. 31 is a partially magnified cross sectional view used to show amethod of manufacturing the device of FIG. 22.

FIG. 32 is a partially magnified cross sectional view used to show amethod of manufacturing the device of FIG. 22.

FIG. 33 is a partially magnified cross sectional view used to show amethod of manufacturing the device of FIG. 22.

FIG. 34 is a partially magnified cross sectional view used to show amethod of manufacturing the device of FIG. 22.

FIG. 35 is a cross sectional view used to show a method of manufacturingthe device of FIG. 22.

FIG. 36 is a partially-magnified cross sectional view showing anextracellular potential measuring device in accordance with a thirdexemplary embodiment.

FIG. 37 is a partially-magnified cross sectional view showing a methodof manufacturing the device of FIG. 36.

FIG. 38 is a partially-magnified cross sectional view showing a methodof manufacturing the device of FIG. 36.

FIG. 39 is a partially-magnified cross sectional view showing anextracellular potential measuring device in accordance with a thirdexemplary embodiment.

FIG. 40 is a cross sectional view showing a typified conventionalextracellular potential measuring device.

DETAILED DESCRIPTION OF INVENTION First Embodiment

FIG. 1 is a partially-cut-off and exploded perspective view of anextracellular potential measuring device in accordance with a firstembodiment; case of which device is partly cut-off in order to show theinside. FIG. 2 is a partially-cut-off perspective view of a sensingelement for measuring extracellular potential (hereinafter referred toas “sensing element”). FIG. 3 is a partially-cut-off and magnifiedperspective view showing a key portion of the sensing element of FIG. 2.

Extracellular potential measuring device (device) 51 is formed of case 2and extracellular potential measuring sensing element 1 attachedthereto. Sensing element 1 is a lamination of silicon and silicondioxide. Case 2 is made of an electrically insulating resin material,and provided with reference electrode 7 for measuring the potentialwithin case 2.

Sensing element 1 is provided with well 9 which have the opening at theupper surface of sensing element 1. At the bottom of well 9, firstmicro-throughhole (throughhole) 3 and depression 4 are provided.Throughhole 3, diameter of which is smaller than that of depression 4,is penetrating through the bottom surface of depression 4 and the lowersurface of sensing element 1. Sensing element 1 is provided at the lowersurface with detective electrode 8, as shown in FIG. 3. Detectiveelectrode 8 is located in a bottom end of throughhole 3.

Case 2 has second throughhole (throughhole) 5 provided at the bottom.Size of throughhole 5 is larger than that of the opening of well 9. Wall10 of well 9 has a bowl shape, and is for keeping culture solution anddrug.

Case 2 is provided in the side wall with third throughhole (throughhole)6. Tube 11 is inserted in throughhole 6. Tube 11 is guided bythroughhole 6 so that a tip-end of tube 11 is accurately positioned onthe place of throughhole 3 and depression 4.

FIG. 5 and FIG. 6 illustrate that the surfaces of depression 4 andthroughhole 3 are covered with oxide layer 46. Oxide layer 46 is formeddepending on needs, in accordance with kind, size, etc. of a subjectcell. Oxide layer 46 varies in the thickness; it may have a thickness of10000 angstrom or more when it is formed through thermal oxidation,while it may have a thickness of 500 angstrom or less when it is formednaturally. The thickness of oxide layer 46 is not an essential conditionto the present invention.

Next, a method how to use measuring device 51 is described.

FIG. 4 is a cross sectional view used to show how to use measuringdevice 51. FIG. 5 and FIG. 6 show magnified cross sectional views ofthroughhole 3 and depression 4.

When culture solution 22 and subject cell (cell) 21 are put into case 2,cell 21 drifts in culture solution 22. Inside of case 2, depression 4and throughhole 3 are filled with culture solution 22, and then culturesolution 22 flows out into the side of detective electrode 8.

Cell 21 drifting in culture solution 22 flows due to a pressure withincase 2, and sucked into depression 4, as shown in FIG. 5 and FIG. 6. Ifa pressure for sucking cell 21 is not high enough, a suction pump (notshown) or the like means is used to pull culture solution 22 from thedetective electrode 8 side. Then, cell 21 drifting in culture solution22 is surely pulled towards depression 4. Since the size of throughhole3 has been designed to be smaller than that of cell 21, cell 21 isretained to be staying within the inside of depression 4.

Meanwhile, in order to have drug (not shown) dispensed in theneighborhood of cell 21 without fail, arranging the tip-end of tube 11needs to be placed as close to depression 4, where cell 21 is held.Since normally-used tube 11 is as fine as 1 mm or less in the diameter,tube 11 can easily be bent due to the surface tension of culturesolution 22 when tube 11 is put into case 2 filled with culture solution22 from the above, as shown in FIG. 7 and FIG. 8. So, it is not an easytask to locate the tip-end of tube 11 accurately at a certain specifiedlocation.

Therefore, throughhole 6 that radiates from a location where throughhole3 and depression 4 are placed is provided in the side wall of case 2.Tube 11 is inserted in throughhole 6. Thus the tip-end of tube 11 can bepositioned accurately to be very close to throughhole 3 and depression4.

As described above, tip-end of tube 11 can be located easily at acertain specified location without being ill-affected by the surfacetension of culture solution 22 which is filling case 2. Namely, the wallsurface of throughhole 6 serves as the guide for tube 11. Thus, drug canbe dispensed at any time in a stable manner.

Since wall 10 is bowl-shaped, the drug can easily be injected into theinside of well 9. Therefore, even if a pressure of dispensing the drugthrough tube 11 is low, the drug can surely make a reach to theneighborhood of cell 21 which is held at depression 4. Cell 21 would notbe stressed inadvertently, and a resultant vibration which could becaused by a pressurized cell 21 can be prevented. The bowl-shaped wall10 functions as a guide section for guiding drug into well 9.

Since throughhole 5 is designed to be larger than the opening of well 9,lower side of case 2 does not cover the bowl contour of well 9. So, thedrug can easily be injected into the bottom part of well 9.

Preferably, tube 11 is made of a material selected from the groupconsisting of a resin, a glass and silica. These materials areelectrically insulating, so insertion of such a material into case 2would not invite a disturbance with the potential of culture solution 22inadvertently.

Next, as shown in FIG. 9, wall 10A of well 9A may be formed in aspiral-shape. The spiral-shape of wall 10A facilitates homogeneousmixing of the dispensed drug with culture solution 22 kept in well 9A,even when a low dispensing pressure is injected for the drug and thespeed of inflow is low. Sensing element 1A having spiral wall 10A isadvantageous in performing high accuracy measurements with a leastspread.

Wall 10B well 9B may be formed instead in a radial pattern which isdirected towards the center, as shown in FIG. 10. The radial pattern ofwall 10B facilitates homogeneous mixing of the dispensed drug withculture solution 22 kept in well 9B, even when a low dispensing pressureis injected for the drug and the speed of inflow is low. Sensing element1B having radial wall 10B offers the same advantages as those offered bythe spiral wall. Spiral-shaped wall 10A, or radial-shaped wall 10B,functions as a guide section for guiding drug into well 9A, or 9B, inthe same manner as the bowl-shaped wall 10.

Size of depression 4 may be changed depending on subject cell 21; anysize would do for the depression 4 in so far as it can retain cell 21within depression 4. Typical dimensions for depression 4 are; 10-500micron for the diameter, 1-500 micron for the depth. 10-100 micron forthe diameter and 2-100 micron for the depth are preferable. Morepreferred dimensions for depression 4 are; 20 micron for the diameterand 10 or 20 micron for the depth.

Size of throughhole 3 may also be changed depending on subject cell 21,like the case with depression 4. Any size would do for the size ofthroughhole 3 in so far as it can retain cell 21 within depression 4.Typical dimensions for throughhole 3 are; 5-100 micron for the diameter,10 nanometer-100 micron for the depth. 5 micron for the diameter and 1.5micron for the depth are preferable.

Now in the following, a method of measuring extracellular potentialusing device 51 is described.

Drug is dispensed via tube 11 into culture solution 22 kept in case 2.The drug permeate within culture solution 22, and to the neighborhood ofcell 21 held at depression 4.

When cell 21 is activated by the drug, the number of times of the ionexchange increases, during which ion exchange cell 21 takes such ions asNa⁺, K⁺, Ca²⁺, etc. in and out through an ion channel within cellmembrane (not shown). The ion exchange is an electrochemical changecaused by cell 21 reacting to the drug.

The increased frequency of ion exchange causes the ion concentrationwithin culture solution 22 to change at the neighborhood of cell 21, orin the inside of throughhole 3, which results in a change in thepotential. Meanwhile, culture solution 22 inside of throughhole 3normally has electrical resistance of 5 mega ohm or higher, and isolatedfrom culture solution 22 kept in case 2. Therefore, change in the ionconcentration or in the potential taking place in the inside ofthroughhole 3 can be picked up by detective electrode 8 in the form ofan electrical signal.

The frequency of ion exchange taking place between cell 21 and culturesolution 22 varies depending on a kind of cell 21. As a general rule,however, phenomenon of the ion exchange is not synchronized with acertain specific cycle, but it is considered that it is performed atrespective ion channels with a certain probability. Namely, theelectrical signals are observed not at a certain specific frequencyalone, but frequency of the detected signals ranges widely, from DClevel to approximately 5 kHz. The electrical signals generated by cell21 have random frequency characteristics.

FIG. 11 shows the flow of an extracellular potential measurement.Electrical signal 62 is detected at detective electrode 8, a sensingsection, as voltage or current. Detected electrical signal 62 isamplified by amplifier 63. Amplified electrical signal 64 is accumulatedat fast Fourier transformer (FFT) 65 for a certain time to performfrequency domain changing. Signal strength 66 at the frequency domain isindicated by computer 67 at a certain specific interval. In this way,electrical signal 62 measured at detective electrode 8 is underwent thefrequency changing, and then indicated and visualized by a computer.

Noise from outside has a regular frequency characteristic. Therefore, anelectrical signal generated by cell 21 can be visually distinguishedfrom the noise through the analysis of signal strength 66 at thefrequency domain. So, even if there are noises coming from externalpower source, etc., the electrical signal generated from cell 21 can beeasily distinguished from the noises, and recognized. Thus, a method ofmeasuring extracellular potential in the present invention can catchaccurately a change in the reaction of cell 21 caused by the drug.

FIG. 12 is a characteristics chart showing measurement data madeavailable through an extracellular potential measuring method. In FIG.12, the horizontal axis exhibits the time of measurement in the unit ofa second, while the vertical axis exhibits the frequency. And, thesignal strengths at the time of measurement are plotted in density(contour chart). Referring to FIG. 12, signal 72 and signal 73 showhorizontal lines. This means that signal 72 and signal 73 have regularfrequency characteristics; namely, these signals are noises coming froman external power supply or the like sources. Signal 71 and signal 74don't have such regular frequency characteristics, which means thatthese signals are the electrical signals generated due to changes in theion concentration in the neighborhood of cell 21.

Next, a method of manufacturing extracellular potential measuring device51 in accordance with a first exemplary embodiment is described. FIG. 13and FIG. 16 through FIG. 20 show cross sectional views of sensingelement 1 used to describe a method of manufacturing extracellularpotential measuring device 51. FIG. 14 and FIG. 15 are magnified crosssectional views showing part of sensing element 1 used to describe amethod of manufacturing extracellular potential measuring device 51.FIG. 21 is a cross sectional view used to describe a method ofmanufacturing extracellular potential measuring device 51.

As FIG. 13 illustrates, substrate 40 has a laminated body formed of base41, intermediate layer 42 and thin plate 43. Base 41 and thin plate 43are made of silicon, while intermediate layer 42 is made of silicondioxide. Resist mask 44 having a certain specific pattern is provided onsubstrate 40 at the surface of thin plate 43 side with. The material forsubstrate 40 is generally called as SOI substrate, which substrate isoften used for manufacturing the semiconductor devices. This means thatthe material is readily available anywhere, so description on the methodof manufacturing substrate 40 is eliminated here.

In the first place, throughhole 3 is formed to thin plate 43 for acertain predetermined depth by dry etching process, as shown in FIG. 14.The most suitable process for providing throughhole 3 is dry etchingprocess. In the dry etching method, a gas to accelerate the etchingeffect (accelerating gas) and a gas to retard the etching effect(retarding gas) are used (not shown). Typical accelerating gases areXeF₂, CF₄, SF₆. Typical retarding gases are CHF₃, C₄F₈. By using amixture of accelerating gas and retarding gas for dry etching, wallsurface of etched hole is covered with a protective layer, which is apolymer of CF₂. The protective layer formed covering the wall surface ofa hole restrains etching against the wall surface. As the result,formation of throughhole 3 proceeds anisotropically only to the downwarddirection of resist mask 44.

The mechanism of anisotropic etching proceeding only in the downwarddirection is described more in detail.

Little etching advance with the acceleration gas in etching step. Andthen, formation of a protective layer by the retarding gas follows toprovide a protective layer a little bit in protective layer formingstep. In this way, the etching action and the formation of protectivelayer are repeated alternately; and a hole is etched in substantiallythe vertical direction and a protective layer is provided to cover thewall surface.

During the etching process, substrate 40 is placed in an atmosphere ofplasma generated by inductive coupling of external coil. And a highfrequency voltage is applied to substrate 40. In the above environment,the negative bias voltage arises in substrate 40, and the positive ionamong the plasma, SF₅ ⁺ or CF₃ ⁺, collides against substrate 40.Thereby, the dry etching proceeds vertically down, and throughhole 3 isformed.

During the formation of protective layer, on the other hand, no highfrequency voltage is applied to substrate 40. Then, no bias voltage isgenerated at substrate 40; so, the positive ion among plasma is notdeflected, and the dry etching is retarded. A fresh new surface appearedas the result of etching action is exposed to the material forprotective layer, CF⁺. Consequently, the wall surface of throughhole 3provided as the result of the dry etching is covered evenly withprotective layer 75. In a dry etching experiment, SF₆ was used for theaccelerating gas, C₄F₈ for the retarding gas, and formation ofthroughhole 3 and protective layer 75 has been confirmed.

As described in the above, when the dry etching procedure with theaccelerating gas and the retarding gas is employed for providingthroughhole 3, formation of the throughhole 3 proceeds in the depthdirection of resist mask 44. Whereas, the retarding gas works to provideprotective layer 75 covering the wall surface of throughhole 3.

Next, as shown in FIG. 15, it is dry etched using only XeF₂, or the likeaccelerating gas. Accelerating gas does not etch protective layer 75formed on the wall surface of throughhole 3; the gas etches only thesilicon surface at the bottom of throughhole 3. Since the accelerationgas does not etch silicon dioxide, intermediate layer 42 is not etched.As the result, depression 4 is formed for a size larger than throughhole3.

Next, as shown in FIG. 16, it is etched using resist mask 45 provided onthe surface at the base 41 side. This etching forms well 9 asillustrated in FIG. 17. The etching process employed for the formationof well 9 is a dry etching which uses only the accelerating gas, e.g.XeF₂, SF₆. Thereby, side wall 10 of well 9 is formed in a bowl shape.

For the purpose of forming well 9 in a spiral shape or a radial shape,dedicated etching masks are used respectively. The etching gas in thiscase contains the retarding gas too, besides the accelerating gas.

Next, as shown in FIG. 18, intermediate layer 42 is etched off by agenerally-used etching process, wet etching process or dry etchingprocess.

And then, resist mask 45 is removed. Further, as shown in FIG. 19, oxidelayer 46 is formed by a generally-used thermal oxidation processcovering the silicon surface. Thermal oxidation process is employed insuch cases where the surface resistance of silicon is requested to be ashigh as possible. In other cases where such a high surface resistance isnot needed, a natural oxide layer formed on the silicon surface isenough.

Next, as shown in FIG. 20, substrate 40 is provided with detectiveelectrode 8 formed on the surface of thin plate 43 side by a sputtering,a vacuum deposition or the like normal thin film formation process,using Au, Al, Ti, Cr, etc.

After processing substrate 40 by various process steps as described inthe above, sensing element 1 is made. The use of a SOI substrate, whichhas a laminated body of silicon and silicon dioxide, for substrate 40makes it possible to process the substrate at a high precision levelthrough significantly simple and easy operations. Thus, sensing element1 can be manufactured efficiently.

Finally, as shown in FIG. 21, sensing element 1 and case 2 are gluedtogether using adhesive 50 or other means. Case 2 can be providedthrough a resin molding or other commonly-used processes; so,description on which is eliminated here. Case 2 should preferably bemade of an electrically-insulating material so that culture solution 22in case 2 does not suffer from an inadvertent change in the potential,and the measurement is not ill-affected.

Reference electrode 7 and tube 11 are inserted for conducting themeasurement.

Although the above descriptions have been based on the use of laminatedsubstrate 40 formed of silicon and silicon dioxide for sensing element1, the material for intermediate layer 42 is not limited to silicondioxide. Those other materials having the higher difficulty of etchingin relation to silicon base 41 and thin plate 43, and provided with ahigh electrically-insulating property, may be used for intermediatelayer 42. A glass material containing silicon dioxide, for example, canbe use for intermediate layer 42.

Second Embodiment

Second exemplary embodiment of the present invention is describedreferring to FIG. 22 through FIG. 27. Those portions having the samestructure as those of the first embodiment are indicated by using thesame symbols. FIG. 22 is a partially cut-off perspective view of anextracellular potential measuring device in accordance with secondembodiment. FIG. 23 is a partially cut-off perspective view of the caseshown in FIG. 22. FIG. 24 is a partially cut-off perspective view of asensing element. FIG. 25 is a magnified perspective view showing thesensing element of FIG. 24, with part of the element cut off.

Referring to FIG. 22, extracellular potential measuring device (device)81 has been structured by attaching sensing element 12 to the bottom ofcase 13. Case 13 is made of an electrically-insulating resin material,and provided with reference electrode 7 which is set in the inside ofthe case for measuring a potential within case 13.

Sensing element 12 has a laminated body of silicon and silicon dioxide.Sensing element 12 has opening section 20 at the bottom part. Detectiveelectrode 19 is placed at a lower surface of sensing element 12. Thelower surface of sensing element 12 is formed by providing openingsection 20. Sensing element 12 is provided at the upper surface withdepression 4. First micro-throughhole (throughhole) 3 is formedpenetrating through the bottom of depression 4 and the lower surface ofsensing element 12 where detective electrode 19 is disposed.

Case 13 has second throughhole (throughhole) 14 at the bottom. Wall 15of throughhole 14 is bowl-shaped. Well 17 is formed by upper surface 16of sensing element 12 which is attached making contact with the bottomof case 13, throughhole 14 and wall 15. A point of difference from thefirst embodiment is in the structure of well 17.

Further, third throughhole 6 is provided in the side wall of case 13. Onthe assumption that sensing element 12 will be attached to the bottom ofcase 13, throughhole 6 is disposed in a radial direction that is comingfrom a certain specific place in the neighborhood of throughhole 3 anddepression 4.

The method of using device 81 and the method of measuring extracellularpotential using the device remain substantially the same as thosemethods in the first embodiment.

The bowl-shaped wall 15 ensures that the drug permeate to theneighborhood of subject cell 21 held in depression 4, even if flow speedfor dispensing drug is slowed. Like in the first embodiment, there willbe no change in the flow of culture solution 22 at the neighborhood ofcell 21. So, the measuring environments can be kept stable. Namely, thebowl-shaped wall 15 works as a guide section for guiding the drug into abottom of well 17.

If wall 23A having a spiral-shape is provided at the bottom of case 13A,as shown in FIG. 26, orientated towards the center of throughhole 14A,drug dispensed into culture solution 22 are mixed homogeneously even ifthe inflow speed of drug is slowed a step further. Thus, wall 23A isadvantageous for performing high accuracy measurements with less spread.

Further, as shown in FIG. 27, if wall 23B having a radial-shapeorientated towards the center of throughhole 14B is provided at thebottom of case 13B, it provides the same advantage for performing thehigh accuracy measurements. Spiral-shaped wall 23A as well asradial-shaped wall 23B work as a guide section for guiding the drug intowell 17, in the same manner the bowl-shaped wall 15 does.

Opening section 20 is not an essential item. In the present embodiment,opening section 20 has been provided for the purpose of producing goodbalance between a thickness of sensing element 12 needed for ensuring asufficient mechanical strength and a depth of throughhole 3. Therefore,opening section 20 is not necessary if a thin sensing element 12 isrigid enough.

Next, a method of manufacturing extracellular potential measuring device81 is described in accordance with second embodiment. FIG. 28 throughFIG. 34 are cross sectional views of sensing element 12 used to show amethod of manufacturing extracellular potential measuring device 81.FIG. 35 is a cross sectional view used to show a method of manufacturingextracellular potential measuring device 81.

As shown in FIG. 28, substrate 25 is formed of base 26, intermediatelayer 27 and thin plate 28. Material for both base 26 and thin plate 28is silicon, while intermediate layer 27 is silicon dioxide. Substrate 25is provided with resist mask 29 formed on the face of base 26 side.Substrate 25 is an SOI substrate, which is often used for manufacturingthe semiconductor devices. This means that the SOI substrate is readilyavailable anywhere, so, the method of manufacturing the substrate iseliminated here.

Base 26 is etched by a generally-used etching process, such as a dryetching or a wet etching, to form opening section 20, as shown in FIG.29.

Next, as shown in FIG. 30, substrate 25 is provided with resist mask 30of a certain specific pattern formed on the surface of thin plate 28.

Then, as shown in FIG. 31, substrate 25 is dry-etched from the face ofthin plate 28 side. The etching is conducted using only the acceleratinggas. The accelerating gas used for the etching is SF₆, CF₄, XeF₂, etc.When silicon is etched by the accelerating gas, the etching proceeds notonly in the direction of depth but it also advances towards widthdirection. This phenomenon has been confirmed in an experimental etchingusing XeF₂. Consequently, as shown in FIG. 31, the etching assumes ahalf-spherical shape concentric with the open area of resist mask 30,and depression 4 is formed. Since resist mask 30 is hardly etched byXeF₂, the mask maintains its original shape.

And then, as shown in FIG. 32, it is etched again from the face of thinplate 28 side, using a mixture of accelerating gas and retarding gas.Thereby, the etching proceeds anisotropically only in thevertically-down direction; so, throughhole 3 is formed starting at thebottom of depression 4.

Next, as shown in FIG. 33, intermediate layer 27 made of silicon dioxideis etched off by a generally-used etching process, such as a wetetching, a dry etching, etc. from base 26 side. After then, resist mask30 is also removed.

Next, as shown in FIG. 34, substrate 25 is provided with detectiveelectrode 19 formed on the face of base 26 side by a generally-used thinfilm forming technology using a material which contains Au as the mainingredient. Sensing element 12 is manufactured through the abovedescribed steps. Depending on needs, thermal oxidation process may beintroduced prior to formation of detective electrode 19 in order to formoxide layer 46 covering the silicon surface.

Then, as shown in FIG. 35, sensing element 12 is glued, with the face ofthin plate 28 side up, to the bottom of case 13 using adhesive 50 orother means, which case has already been manufactured separately with aresin material. Case 13 can be manufactured by a normally-used meanssuch as a press molding, formation with optical means, cutting andmachining, etc. Throughhole 14 at the bottom and throughhole 6 in theside wall of case 13 can be formed to a desired shape by making use ofthese technologies. There is no specific difficulty in the formation ofthroughhole 14 and throughhole 6; so, no description is made here onthis point.

Extracellular potential measuring device 81 in the present secondembodiment is thus manufactured.

Although the above descriptions have been based on the use of laminatedsubstrate 25 formed of silicon and silicon dioxide for the manufactureof sensing element 1, the material for intermediate layer 27 is notlimited to silicon dioxide. Other materials which have the higherdifficulty of etching relative to silicon base 26 and thin plate 28, andare provided with a property of high electrical insulation may be usedfor intermediate layer 27. A glass material containing silicon dioxide,for example, can be use for intermediate layer 27.

Third Embodiment

Third exemplary embodiment of the present invention is describedreferring to FIG. 36 through FIG. 38. Those portions having the samestructure as those of the first embodiment are indicated by using thesame symbols.

FIG. 36 is a cross sectional view showing the structure of anextracellular potential measuring device in accordance with thirdembodiment.

Those disclosed in the present third embodiment are applicable to theextracellular potential measuring device of first embodiment or secondembodiment, and provide the same advantages.

The point of difference in third embodiment as compared with the firstembodiment is that, as shown in FIG. 36 and FIG. 38, part of detectiveelectrode 8 on sensing element 38 is covered with insulating layer 31.As the result, detective electrode 8 is exposed only at an area in thevicinity of throughhole 3. The exposed area of detective electrode 8 isopening section 37. Furthermore, insulating substrate 39 made of a glassmaterial is provided to cover detective electrode 8 with a certain gapin between.

The procedure of detecting extracellular potential remains the same asin the first embodiment. So, no description is made on this respect.Here, the advantage of having insulating layer 31 on the surface ofdetective electrode 8 and the advantage of having insulating substrate39 are described.

As already described in the first embodiment referring to FIG. 4 andFIG. 5, culture solution 22 flows out also into the detective electrode8 side. Then, the flown out culture solution 22 makes contact withdetective electrode 8 at the exposed area.

A region in which the ion concentration or the potential changes due toion exchange of cell 21 is limited only to the region that is very closeto cell 21. That is to say, the potential changes are limited only atthe neighborhood of throughhole 3. However, the change in potential ofculture solution 22 detected by detective electrode 8 is not limited tothat taking place at the neighborhood of throughhole 3. For example,suppose the potential has changed in a region away from throughhole 3(e.g. circled area C in FIG. 4) due to a noise which is irrelevant toactivity of cell 21, detective electrode 8 detects the change ofpotential caused by the noise, either. Then, an electrical signaldetected by detective electrode 8 represents the average of potentialchange due to ion exchange by cell 21 and that caused by noise. As theresult, it is difficult to detect accurately only the potential changedue to ion exchange by cell 21.

Therefore, as shown in FIG. 36, part of detective electrode 8 is coveredwith insulating layer 31, leaving only the limited area very close toopening section 37 exposed. Thereby, detective electrode 8 does notdetect a change in the potential due to noise (e.g. a change takingplace in the circled area D in FIG. 36). Thus the device yields stablemeasurement data without being affected by disturbing factors.

The smaller the exposed area of detective electrode 8, the less it isaffected by noise; and stable measurement data are made available. Forobtaining stable measurement results, an area of detective electrode 8covered with insulating layer 31 should preferably be larger than anexposed area of detective electrode 8.

Furthermore, insulating substrate 39 suppresses the fluctuation withculture solution 22 at the detective electrode 8 side; hence, it reducesnoise due to the fluctuation with culture solution 22. Namely, in device51 of FIG. 4, culture solution 22 at the detective electrode 8 side isexposed to the atmospheric air. So, if it is affected by an externalvibration or if the atmospheric pressure changed during a measuringoperation, culture solution 22 may sometimes cause fluctuation at thesurface. The fluctuation at the surface generates a stream withinculture solution 22. Then, a stream is generated also in culturesolution 22 at the neighborhood of throughhole 3. This can lead to anoise. Therefore, it is difficult for detective electrode 8 to measureaccurately the change in the ion concentration or in the potential dueto ion exchange by cell 21.

Still further, a change of pressure occurred at the neighborhood ofthroughhole 3 may sometimes give influence to a pressure of holding cell21. When cell 21 encounters a pressure change, an electrical insulationresistance separating throughhole 3 from case 2 also changes. At thesame time, cell 21 changes its own state of electrical activity whencell 21 is exposed to a pressure change. Electrical signal detected atdetective electrode 8 changes depending on a change in the insulationresistance or a change in cell 21's own state of electrical activity. Asthe result, it is difficult to measure accurately a change of thepotential due to ion exchange which is caused by cell 21 reacting to thedrug.

In the present embodiment, insulating substrate 39 is provided makingcontact to the surface of detective electrode 8 with a certain gap inbetween, as shown in FIG. 36. Culture solution 22 flown out into thedetective electrode 8 side is confined between sensing element 38 andinsulating substrate 39; therefore, the culture solution 22 is notexposed directly to the atmospheric air. So, it is least influenced byexternal forces. Even if an external vibration or a change in theatmospheric pressure takes place during a measuring operation,fluctuation will not arise with the surface of culture solution 22.

Thus, measuring device 91 detects extracellular potential accuratelywith a good stability. In order not to bring the potential of culturesolution 22 into an inadvertent instability, it is preferred thatinsulating substrate 39 is made of an electrically-insulating material.

Now in the following, a method of manufacturing device 91 is describedin accordance with third embodiment. Since the present third embodimentuses substrate 40 whose manufacturing method has already been describedin the first embodiment, the manufacturing process steps described inthe first embodiment (FIG. 13-FIG. 20) are not repeated here.

In the first place, substrate 40 as shown in FIG. 20 is prepared, whichhas been already provided with detective electrode 8 formed thereon.Then, as shown in FIG. 36, substrate 40 is provided with insulatinglayer 31 formed on the face of detective electrode 8 side, with anelectrically-insulating material sputtered, vacuum deposited orspin-coated thereon, or through other processes. Polyimide, fluororesin,silicon dioxide, etc. may be used for the electrically-insulatingmaterial.

Next, as shown in FIG. 38, unnecessary part of insulating layer 31 isetched off using a resist mask, or by other suitable method, to forminsulating layer 31 in a certain specific pattern. Thus sensing element38 is obtained, detective electrode 8 of which being exposed only in alimited area at the neighborhood of throughhole 3.

Then, sensing element 38 is glued, with the face of detective electrode8 side down, to the bottom of case 2, using adhesive 50 or other means.And then, as shown in FIG. 36, insulating substrate 39 is glued tosensing element 38 at the face of detective electrode 8 side, with acertain specific gap secured in between. In order to ensure a highaccuracy of measurement, it is preferred to use an insulating resinadhesive (not shown) for gluing insulating substrate 39.

The above-described configuration in third embodiment can be appliedalso to device 81 of the second embodiment. FIG. 39 is apartially-magnified cross sectional view showing extracellular potentialmeasuring device 81 of the second embodiment, on which the configurationof third embodiment is applied.

The point of difference as compared with the second embodiment is that,as shown in FIG. 39, part of detective electrode 19 on sensing element58 is covered with insulating layer 31. As the result, detectiveelectrode 19 is exposed only at an area that is close to throughhole 3'sopening section 37. Furthermore, insulating substrate 39 made of a glassmaterial covers detective electrode 19 with a certain gap in between.The device exhibits the same functioning and advantages as described inthe above.

In manufacturing extracellular potential measuring device 92, the samemanufacturing method used for device 91 can be used.

1. A device for measuring extracellular potential comprising: a sensingelement including: a substrate having an upper surface and a lowersurface; a detective electrode provided below the substrate, wherein adepression and a first throughhole penetrating through the depressionand the lower surface are provided at the substrate; and a well which iswider than said depression and which is formed on a top of thedepression by the upper surface of the substrate; a case made of aninsulating material and having a second throughhole at a bottom of thecase, wherein the bottom of the case is attached to the upper surface ofthe sensing element so that the second throughhole is in communicationwith said first throughhole.
 2. A device for measuring extracellularpotential according to claim 1, wherein the case is provided with athird throughhole penetrating through the case in a radial directionsuch that the third throughhole penetrates to the well to expose thedepression, the case further comprising: a tube inserted in the thirdthroughhole, provided for dispensing either the culture solution or thedrug into the case.
 3. A device for measuring extracellular potentialaccording to claim 2, wherein the tube is made of one material of aresin, a glass and silica.
 4. A device for measuring extracellularpotential according to claim 1 further comprising: an insulating layerfor covering part of the detective electrode.
 5. A device for measuringextracellular potential according to claim 4, wherein an area of thedetective electrode covered by the insulating layer is greater than anexposed area of the detective electrode.
 6. A device for measuringextracellular potential according to claim 1 further comprising: aninsulating substrate disposed opposed to the detective electrode with agap in between.
 7. A device for measuring extracellular potentialaccording to claim 1, wherein the substrate has a laminated body formedof, silicon; and one material of silicon dioxide and a glass materialcontaining silicon dioxide.